Project Summary Over half of sports injuries involve tendons and entheses, the insertion sites of tendons or ligaments to bones. A common enthesis disorder enthesophathy, representing one fourth of tendon diseases, results in a long-term disturbance of the load transfer and is a major cause of pain and disability. The exact pathogenesis of enthesopathy is largely unknown, and there is no effective disease modifying treatment for this disorder. The proposed project aims to elucidate the pathogenic mechanisms of enthesopathy and develop potential therapeutic solutions to prevent/treat this disease. Our preliminary studies suggest that TGF? activation in bone tissue of the enthesis, in response to aberrant mechanical loading after enthesis injury, is an early change in the progression of enthesopathy. In a mouse model of enthesopathy, we observed upregulated TGF? activity at the bone-fibrocartilage junction earlier, and entheses progressive degradation later after entheses injury. Moreover, transgenic mice with active TGF? overexpression in bone recapitulated the enthesopathy phenotype, whereas the enthesopathy mice treated with a TGF? neutralizing antibody had decelerated enthesis degeneration. To achieve bone-targeting delivery and slow release in bone tissue, we generated a new drug that conjugates a TGF? inhibitor to Alendronate through a metabolically hydrolysable linker. The conjugate effectively inhibited TGF? activity in bone in the active TGF? transgenic mice. The central hypothesis of this study is that aberrant TGF? activation at the bone-fibrocartilage junction initiates enthesopathy by recruiting stem/progenitor cells for blood vessel invasion and ossification in the fibrocartilage zone. To test this hypothesis, we will examine the role of elevated active TGF? in the progression of enthesopathy using different mouse models (Aim 1). We will also determine the role of TGF?-recruited stem/progenitor cells in the progression of enthesopathy using two genetic lineage tracing mice and an inducible tissue-specific TGF? receptor-ablation mice (Aim 2). Finally, we will examine the therapeutic potential of the TGF? inhibitor?Alendronate conjugate in enthesopathy (Aim 3). Results will provide evidence for clinical application of this conjugate as a therapy for enthesopathy.